Richard H. Sanger

Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.

Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux

Biological systems have very different internal ion compositions in comparison with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasma membrane, we can classify these mechanisms into three types, pumps, porters, and channels. Channels have been extensively studied, particularly since the advent of the patch clamp technique, which opened new windows into ion channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca2+‐ATPase, and porters are more illusive. The technique described in this paper, the self‐referencing, ion‐selective (or Seris) probe, has the ability to monitor the behavior of membrane transport mechanisms, such as the pumps and porters, in near to real‐time by non‐invasively measuring local extracellular ion gradients with high sensitivity and square micron spatial resolution.

Imaging the electric field associated with mouse and human skin wounds

We have developed a noninvasive instrument called the bioelectric field imager (BFI) for mapping the electric field between the epidermis and the stratum corneum near wounds in both mouse and human skin. Rather than touching the skin, the BFI vibrates a small metal probe with a displacement of 180 μm in air above the skin to detect the surface potential of the epidermis through capacitative coupling. Here we describe our first application of the BFI measuring the electric field between the stratum corneum and epidermis at the margin of skin wounds in mice. We measured an electric field of 177±14 (61) mV/mm immediately upon wounding and the field lines pointed away from the wound in all directions around it. Because the wound current flows immediately upon wounding, this is the first signal indicating skin damage. This electric field is generated at the outer surface of the epidermis by the outward flow of the current of injury. An equal and opposite current must flow within the multilayered epidermis to generate an intraepidermal field with the negative pole at the wound site. Because the current flowing within the multilayered epidermis is spread over a larger area, the current density and subsequent E field generated in that region is expected to be smaller than that measured by the BFI beneath the stratum corneum. The field beneath the stratum corneum typically remained in the 150–200 mV/mm range for 3 days and then began to decline over the next few days, falling to zero once wound healing was complete. The mean wound field strength decreased by 64±7% following the application of the sodium channel blocker, amiloride, to the skin near the wound and increased by 82±21% following the application of the Cl− channel activator, prostaglandin E2.

Intracellular Release of Caged Calcium in Skate Horizontal Cells Using Fine Optical Fibers

Horizontal cells are second order retinal neurons that receive direct input from photoreceptors and are involved in establishing a number of key features of visual perception. These cells mediate the formation of the inhibitory surround portion of the classic center-surround receptive fi elds of retinal neurons (1). The centersurround receptive fi elds are important for enhancing the contrast of visual objects and are also involved in color perception. The molecular mechanisms by which horizontal cells send lateral inhibitory signals to photoreceptors and bipolar cells are still under debate, but protons released from horizontal cells have been hypothesized to alter the fl ow of visual information within the outer retina (2). Indeed, small changes in extracellular pH can dramatically alter neural signals within the retina, in part because photoreceptor calcium channels are highly sensitive to protons. When protons bind to photoreceptor calcium channels, the voltage activation range of the channels shifts to more depolarized potentials and the overall conductance of the cell to calcium is reduced, which signifi cantly reduces neurotransmitter release (3). Our previous work has shown that glutamate, the neurotransmitter released by photoreceptors onto horizontal cells, modulates the fl ux of hydrogen ions from skate retinal horizontal cells (4). Glutamateinduced changes in H fl ux depend on the presence of extracellular calcium and likely refl ect the activation of plasma membrane calcium/H ATPases. These transporters extrude intracellular cal cium in exchange for extracellular hydrogen ions, decreasing the concentration of protons at the extracellular face of the horizontal cells (5).